ES2704002T3 - Method to produce an acidified milk product - Google Patents

Abstract

A method of producing a yogurt with decreased stringiness, wherein said method comprises: a) providing a milk substrate; b) treating the milk substrate with an enzyme having transglutaminase activity; and c) fermenting the milk substrate with a starter culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce yogurt, where said starter culture produces an exopolysaccharide (EPS).

Description

DESCRIPTION

Method to produce an acidified milk product

Technical field

The present invention relates to a method for producing a yogurt with improved texture and pleasant mouthfeel.

BACKGROUND OF THE INVENTION

The market for acidified milk products, which includes fermented milks, such as yogurt that can be taken with a spoon, firm and liquid (drinkable), is increasing worldwide and there is an interest in improving the quality and economy of this product. .

Acidified milk products are generally produced by the acidification of a milk base, such as fresh pasteurized milk or reconstituted milk from powdered milk protein and water. The acidification can take place by the addition of a chemical substance, such as glucono delta-lactone (GDL) or lactobionic acid (LBA), or it can be produced by the fermentation of milk with lactic acid bacteria. Optionally, the acidified milk product is mixed with a sugar syrup solution, and / or subjected to a homogenization treatment.

Transglutaminase (TGase or TG) can be used to increase the viscosity and gel firmness of fermented milk products, such as, for example, yogurt. While TGase significantly increases the gel firmness of yogurt, the thickness in the mouth of yogurt tends to become watery and light.

Lauber S et al. (2000), European Food Research and Technology, vol. 210, no. 5, pages 305-309 teaches the effect of transglutaminase in yoghurts to increase its resistance to breakage.

Bonisch et al. (2007), Food Hydrocolloids, vol. 21, no. 4, pages 585-595 and Bonisch et al. (2007), International Dairy Journal, vol. 17, no. 11, pages 1360-137 teach the effect of transglutaminase in yoghurts to increase viscosity and firmness.

EP 0610549 A1 teaches the use of transglutaminase to prevent syneresis in yoghurts.

Lorenzen P C et al. (2005), Kieler Wirtschaftliche Forschungsberichte, vol. 57, no. 2, pages 97-115 teaches the use of transglutaminase in yoghurts to increase viscosity and reduce syneresis.

EP 2011 402 A1 teaches the use of transglutaminase to reduce syneresis and increase the richness sensation of yoghurts.

WO 2009/010257 A2 teaches the use of transglutaminase in yoghurt beverages.

WO 2007/060288 A1 discloses the use of transglutaminase for the production of the Scandinavian milk product viili.

It is an object of the present invention to provide a method for the manufacture of a yogurt with decreased fibrosity compared to a standard acidified milk product.

Compendium of the invention

The present inventors have observed that when yogurt is produced using a bacterial culture of lactic acid that produces exopolysaccharide (EPS) in combination with TGase, it is possible to produce a yoghurt with high gel firmness, as well as a nice thick mouthfeel and a feeling in the mouth nice.

Surprisingly, it has been found that using a combination of a culture that produces EPS (which normally provides a fibrous texture to yogurt) together with TGase, it is possible to produce a yoghurt with a high gel firmness, high thickness in the mouth and a firm texture, more than the fibrous texture normally induced when only the starter culture is used.

Therefore, the present inventors have surprisingly found that a yogurt produced with transglutaminase and a microorganism producing polysaccharides has, for example, improved mouthfeel and reduced fibrosity compared to a yogurt produced in the same manner, but without the transglutaminase treatment.

Accordingly, The present invention relates to a method for producing a yogurt with decreased fibrosity, said method comprising:

a) providing a milk substrate;

b) treating the milk substrate with an enzyme having transglutaminase activity; Y

c) fermenting the milk substrate with a starter culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce the yogurt, wherein said starter culture produces an exopolysaccharide (EPS).

The method of the invention will decrease the fibrousness of a yogurt, and improve the texture and / or the thickness in the mouth and / or the mouthfeel of a yogurt, thereby producing a product that has a pleasant mouthfeel.

In another aspect, the present invention relates to the use of transglutaminase in combination with a microorganism that produces an exopolysaccharide (EPS) to decrease the fibrosity of an acidified milk product, such as a yogurt, for example, a firm-type yogurt or which can be taken with a spoon.

Detailed disclosure of the invention

In its broadest aspect, the present invention relates to a method for producing a yogurt with decreased fibrosity, said method comprising:

a) providing a milk substrate;

b) treating the milk substrate with an enzyme having transglutaminase activity; Y

c) fermenting the milk substrate with a starter culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce the yogurt, wherein said starter culture produces an exopolysaccharide (EPS).

Interesting embodiments of the method of the invention are:

- a method, wherein the yogurt is produced substantially without, or completely without any addition of a thickener and / or stabilizer, such as pectin, gelatin, starch, modified starch, carrageenan, alginate, and guar gum.

- a method, wherein step b) is carried out before or during step c).

- a method, wherein the milk substrate is pasteurized before acidification and the enzyme treatment is carried out before pasteurization.

- a method, wherein the milk substrate is subjected to a heat treatment before treatment with the enzyme having transglutaminase activity.

- a method, wherein glutathione is added to the milk substrate before treatment with the enzyme having transglutaminase activity.

- a method, wherein a protein hydrolyzate is added to the milk substrate before treatment with the enzyme having transglutaminase activity.

- a method, wherein the protein hydrolyzate is selected from the group consisting of: a milk protein hydrolyzate (such as casein hydrolyzate, Nz Case or Maxicurd); a peptone (such as potato peptone); a yeast extract; and a hydrolysed yeast extract.

- a method, wherein the yogurt has a non-fat solid content of milk of less than 8%.

- a method, where the yogurt has a fat content of less than 2%.

- a method, wherein the yogurt has a fat content of less than 0.5%.

- a method, wherein the enzyme having transglutaminase activity is produced recombinantly. - a method, wherein the enzyme having transglutaminase activity is obtained from a bacterium belonging to the genus Streptomyces.

An acidified milk product obtainable by the method of the invention is also disclosed. The product may be packaged, for example, in a sealed container having a volume in the range of 25 to 1500 ml. The acidified milk product is free, or substantially free, of stabilizers and thickeners such as HM pectin, LM pectin, starch, modified starch, gelatin, CMC, soybean fiber / soybean polymer, alginate. By "substantially free" it is to be understood that the product comprises less than 20% (w / w) (eg, less than 10%, less than 5% or even less than 2% or 1%) of stabilizers or thickeners.

In a further aspect, the invention relates to the use of transglutaminase in combination with a microorganism that produces an exopolysaccharide (EPS) to decrease the fibrosity of an acidified milk product, such as a yogurt, for example, a firm-type yogurt or which can be taken with a spoon.

Interesting embodiments of this invention are:

- the use of transglutaminase to decrease the fibrosity of an acidified milk product that has been produced using a microorganism that produces a viscous / fibrous texture in the acidified milk product such as a firm-type yogurt or that can be taken with a spoon.

The terms "fibrous" or "fibrous" refer to the texture of the yogurt and / or acidified milk product. For example, the texture of a yogurt is evaluated by sensory evaluation. A spoonful of yogurt is removed from the sample and the property of fibrous is evaluated. The longer the strand becomes before it breaks, the more fibrous the product is. Fibrousness can also be evaluated instrumentally: using a reometer with a concentric cylinder system, a flow curve measures the shear stress as a function of shear rates from 10 to 350 s-1 (sweeps up and down). The area of the hysteresis loop between the up and down flow curve (shear rates from 0 to 241 s-1) was calculated in% of the area under the upper curve. The area of the hysteresis loop correlates with perceived sensory fibrosity.

The term "firm texture" refers to the opposite of fibrous texture. So when the fibrousness of a yogurt decreases, the texture becomes "firmer".

The term "acidified milk products" refers to any product based on milk that has been acidified, and includes fermented milk products, and acidified milk beverages.

The term "yogurt" covers a milk product produced by fermentation by a starter culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus where the starter culture produces an exopolysaccharide (EPS).

"Acidified milk beverages" include any drinkable product based on acidified milk substrates, which therefore includes fermented milk beverages and liquid yoghurt beverages. Acidified milk drinks are drinkable in the sense that they are in liquid form and are consumed as beverages, that is, they are suitable for drinking instead of being eaten with a spoon ("thick"). "In liquid form" means that the products are in the fluid state of matter thus showing a characteristic disposition to flow. Therefore, the shape of a liquid is usually determined by the filling container, unlike, for example, a gel-like substance, which is soft, but does not flow freely, such as, for example, yogurt or pudding. The acidified milk beverages according to the invention can have a viscosity that allows the consumer to drink the products using a straw if desired.

In an interesting aspect, the acidified milk beverages have a viscosity measured as discharge time from a 10 ml pipette which is substantially the same as the discharge time of an acidified milk beverage produced without transglutaminase. In this context, a discharge time which is substantially the same means that it is increased less than 20%, preferably increased less than 15% and more preferably increased less than 10%.

An acidified milk product can have a pH of less than 4.6, preferably less than 4.4, more preferably less than 4.2, and even more preferably about pH 4.0 or less. In one aspect, the acidified milk product has a pH of less than 3.8, such as less than 3.6.

The acidification is carried out as a fermentation with a microorganism and / or by the addition of an acid, such as an organic acid (for example, lactic acid, lactobionic acid or GDL). The milk substrate is acidified by fermentation with a microorganism. Optionally, such acidification by fermentation is combined with chemical acidification of the milk substrate, for example, by addition of an acid as described above.

An acidified milk product can have a fat content of 0 to 2%, preferably below 1.5%, below 1% or below 0.5%, more preferably of about 0.1% or less. The acidified milk product can have a non-fat solid milk content of less than 20%, preferably less than 8.5%, less than 8%, less than 7.5%, less than 7%, less than 6.5 % or less than 6%, and more preferably about 5%.

An acidified milk product can have a protein content of between 0.5 and 4%. In a preferred aspect, the acidified milk product has a protein content of below 1%. In another preferred aspect, the acidified milk product has a protein content of between 2% and 3%.

An acidified milk product can have a shelf life of more than 7 days, preferably more than 14 days, more preferably more than 28 days, such as more than 3 months. The term "period of validity" as used in this document should be understood as the period of time from the end of a product and until this product, when stored properly and under the conditions recommended by the manufacturer, is It becomes unacceptable to the consumer.

Sensation in the mouth is the physical and chemical interaction of a product in the mouth, an aspect of food rheology. It is evaluated from the initial perception on the palate, through swallowing to the aftertaste. The term describes all tactile observations related to texture and texture sensation in the mouth (or sensations that occur in the oral cavity, related to oral tissues and their perceived state, for example, coating), including the characteristic "creaminess""Which usually refers to the sensation in the mouth of the cream. (Barnes et al., 1991, Journal of Dairy Science 74: 2089-2099, Lawless and Heyman (1999) Sensory evaluation of food: principles and practices, Aspen Publishers, Inc., Gaithersburg, MD).

"Thickness in the mouth" can be described as the degree of thickness when the yogurt is swallowed at a normal-high food speed; the high thickness in the mouth corresponds to a thick product that takes time to swallow (Meilgaard, M., Civille, V.G., Carr, B.T., eds., 1999. Sensory Evaluation Techniques (3rd Edition), New York: CRC Press.).

The term "thick" should be understood as to be consumed using a spoon. The term "fermented milk product that can be taken with a spoon" includes "whipped yogurt". The term "whipped yogurt" refers specifically to a yogurt product that sustains a mechanical treatment after fermentation, which produces a softening and liquefaction of the clot formed in the fermentation stage. The mechanical treatment typically, but not exclusively, is obtained by agitation, pumping, filtration or homogenization of the yogurt gel, or by mixing it with other ingredients. Shake yogurts typically, but not exclusively, have a non-fat solid milk content of 9 to 15%. The term "firm-type fermented milk product" includes a milk-based product that has been inoculated with a starter culture, for example, a yogurt starter culture, and packaged after the inoculation step and then fermented in the package. The term "drinkable fermented milk product", "acidified milk drink" and "fermented milk drink" includes beverages such as "drinking yogurt" and the like. The term "drinking yoghurt" typically covers a milk product produced by fermentation by the combination of a species of Lactobacillus (e.g., L. bulgaricus) and Streptococcus thermophilus. "Yogurt for drinking" is typically consumed by drinking the yogurt, for example, directly from the container or from a glass / cup or the like. Yogurt for drinking typically has a non-fat solid milk content of 8% or more. In addition, the live culture count for drinking yoghurt beverages is typically at least 10E6 cell-forming units (CFU) per ml.

"Milk substrate", in the context of the present invention, can be any raw and / or processed milk material that can be subjected to acidification according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk-like products comprising protein, such as whole or semi-skimmed milk, skimmed milk, buttermilk, reconstituted milk powder, condensed milk, dry milk, whey, whey filtrate, lactose, mother liquor of lactose crystallization, whey protein concentrate, or cream. Obviously, the milk substrate can be milk. The term "milk" should be understood as the milk secretion obtained by milking any mammal, such as cows, sheep, goats, buffalo or camels. In a preferred embodiment, the milk is cow's milk.

In one aspect of the present invention, the milk substrate is more concentrated than the raw milk, i.e., the protein content is higher than in the raw milk. In this aspect, the protein content is more than 5%, preferably more than 6%, such as more than 7%, more preferably more than 8%, such as more than 9% or more than 10%. Preferably, the lactose content is also higher than in raw milk, such as more than 7%, more than 8%, more than 9%, more than 10%, more than 11% or more than 12%. In a preferred embodiment of this aspect, the milk substrate is a concentrated aqueous solution of skimmed milk powder having a protein content of more than 5% and a lactose content of more than 7%.

In the context of the present invention, the percentages defining the content of the milk substrate or the content of the acidified milk product are percentages by mass, i.e. the mass of a substance (eg, protein or lactose) as a percentage of the mass of the whole solution (milk substrate or acidified milk product). Thus, in a milk substrate having a protein content of more than 5%, the mass of the proteins constitutes more than 5% of the mass of the milk substrate.

Preferably, at least part of the protein in the milk substrate is natural protein in milk, such as casein or whey protein. However, part of the protein may be unnatural proteins in milk.

The term "hydrolyzate" refers to any substance produced by hydrolysis. The term is not intended to be limited to substances produced by any specific method of hydrolysis. The term is intended to include "hydrolysates" produced by enzymatic as well as non-enzymatic reactions. For example, any of the known hydrolytic enzymes (eg, proteases, serine proteases, metalloproteases, hydrolases, etc.) are capable of producing hydrolysates within the meaning of how the term is used in the present context. Similarly, non-enzymatic hydrolysis methods (eg, acid / base hydrolysis, etc.) also produce hydrolysates within the meaning of how the term is used in the present application. The term "protein hydrolyzate" refers to a hydrolyzate produced by hydrolysis of a protein of any type or class. Any known protein can be hydrolyzed to produce a protein hydrolyzate within the meaning of the term. A "protein hydrolyzate" can be produced by enzymatic as well as non-enzymatic methods and can include protein fragments (e.g., polypeptides) ranging in size from two to 100 or more amino acids. In addition, as used herein, a "protein hydrolyzate" is not limited to a single product compound, but may include a heterogeneous distribution or mixture of hydrolysis products (e.g., protein fragments). It may also include a homogeneous compound or purified fraction of hydrolysis products. The term "protein" refers to any composition composed of amino acids and recognized as a protein by those skilled in the art. The terms "protein", "peptide", and "polypeptide" are used interchangeably herein. The term "milk protein hydrolyzate" refers to a hydrolyzate produced by hydrolysis of a milk protein of any type or class, for example, a casein or a whey protein.

The proteases usable in the present invention are in particular proteases of class EC 3.4.-.- of the Nomenclature of Enzymes of the IUBMB, especially of the subclasses EC 3.4.21-, EC 3.4.22.-, EC 3.4.23 .- and EC 3.4.24.-. Classes include serine proteases, Bacillus proteases, cysteine proteases, aspartic proteases, metalloproteases, proteases classified in Ec 3.4.21.62, EC 3.4.22.2, EC 3.4.23.4, EC 3.4.24.28, Neutrase®, Alcalase®, subtilisin A ( type VIII), papain, chymosin, Colorase N, Optimase or Protease N "Amano". The protease can be used in purified form, for example, isolated from a microorganism, such as a protease originating from a lactic acid bacterium, or it can be produced in situ by a microorganism, such as the lactic acid bacteria used for fermentation. .

Prior to fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.

"Homogenization" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If the homogenization is carried out before the fermentation, it can be carried out in such a way that the fat of the milk is broken into smaller sizes so that it does not separate more from the milk. This can be achieved by forcing milk at high pressure through small holes.

"Pasteurization" as used herein means treating the milk substrate to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. The specified temperature is usually achieved by heating. The temperature and duration can be selected to destroy or inactivate certain bacteria, such as harmful bacteria. You can follow a quick cooling stage.

"Fermentation" in the methods of the present invention means the conversion of carbohydrates to alcohols or acids by the action of a microorganism. Preferably, the fermentation in the methods of the invention comprises the conversion of lactose to lactic acid.

"Microorganism" can include any bacteria or fungus that is capable of fermenting the milk substrate. The microorganisms used for most fermented milk products are selected from the group of bacteria generally termed lactic acid bacteria. As used herein, the term "lactic acid bacterium" designates a gram positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid such as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are in the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp., and Propionibacterium spp. In addition, lactic acid producing bacteria belonging to the group of strict anaerobic bacteria, bifidobacteria, ie, Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are often used as food crops alone or in combination with other lactic acid bacteria. Lactic acid bacteria are normally supplied to the dairy industry either as frozen or lyophilized cultures for mass initiation propagation or as the so-called "Direct Vat Set" (DVS) cultures, which are intended for direct inoculation into a container or tub of fermentation for the production of a dairy product, such as an acidified milk product. Such crops are generally referred to as "starter cultures" or "starters."

Strains of commonly used starter cultures of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30 ° C and thermophilic organisms having optimum growth temperatures in the range of about 40 ° C to about 45 ° C. ° C. Typical organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp. paracasei The thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

Also, strict anaerobic bacteria belonging to the genus Bifidobacterium including Bifidobacterium bifidum and Bifidobacterium longum are commonly used as dairy starter cultures and are generally included in the group of lactic acid bacteria. In addition, Propionibacteria species are used as dairy starter cultures, in particular for the manufacture of cheese. In addition, organisms belonging to the genus Brevibacterium are commonly used as food starter cultures.

Another group of microbial starter cultures are fungal cultures, including yeast cultures and filamentous fungal cultures, which are used particularly in the manufacture of certain types of cheese and beverages. Examples of fungi include Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae .

In the present invention, the microorganism used for the fermentation of the milk substrate is a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Optionally, the fermented milk substrate can be subjected to heat treatment to inactivate the microorganism.

The fermentation processes that are to be used in the production of acidified milk products are well known and the person skilled in the art knows how to select the appropriate process conditions, such as temperature, oxygen, amount and characteristics of microorganism (s) and processing time. Obviously, the fermentation conditions are selected so as to support the achievement of the present invention, that is, to obtain a suitable fermented milk product in the production of an acidified milk drink.

Also, the person skilled in the art will know how and when additives such as, for example, carbohydrates, flavorings, minerals, enzymes (for example, rennet, lactase and / or phospholipase) have to be used in the production of yoghurt according to the invention.

Optionally, the fermented milk substrate can be diluted to obtain the acidified milk beverage. In one embodiment, the fermented milk substrate is diluted at least 1.5 times, preferably at least 2 times, at least 2.5 times or at least 3 times. It can be diluted with water or an aqueous solution of any kind. "Diluted at least 1.5 times" in the context of the present invention means that the fermented milk substrate is diluted so that its volume increases by at least 50%.

In one embodiment, a syrup is added to the fermented milk substrate. "Syrup" in the context of the present invention is any additional additive ingredient that imparts flavor and / or sweetness to the final product, i.e. yogurt. It may be a solution comprising, for example, sugar, sucrose, glucose, liquid fructose sugar, aspartame, sugar alcohol, fruit concentrate, orange juice, strawberry juice and / or lemon juice. The mixture of the fermented milk substrate and the syrup can be homogenized using any method known in the art. The homogenization can be carried out in such a way that a homogeneous liquid solution is obtained which is smooth and stable. The homogenization of the mixture of the acidified milk substrate and the syrup can be carried out by any method known in the art, such as forcing milk at high pressure through small holes.

In another embodiment of the invention, water is added to the fermented milk substrate, and the mixture of fermented milk substrate and water is homogenized.

The methods of the present invention comprise treating the milk substrate with an enzyme having transglutaminase activity. The enzyme treatment can be carried out before fermentation, such as before inoculation with the microorganism. The enzyme treatment can be carried out at the same time as the fermentation. In one embodiment, the enzyme is added before, at the same time or after inoculation of the milk substrate with a microorganism, and the enzymatic reaction in the milk substrate occurs essentially at the same time as it is fermented. Alternatively, the enzymatic treatment can be carried out after fermentation. If the acidified milk substrate is mixed and optionally homogenized with the syrup, the enzymatic treatment can be carried out before or after this. The enzyme can be added at the same time or after the syrup, but before homogenization, or it can be added after the acidified milk substrate and the syrup have been mixed and homogenized.

In a preferred embodiment, the enzymatic treatment is carried out before or during fermentation. In a more preferred embodiment, the milk substrate is pasteurized prior to fermentation, and the enzymatic treatment is carried out before pasteurization. Pasteurization can, therefore, inactivate the enzyme.

In another preferred embodiment, the milk substrate is subjected to heat treatment, such as pasteurization, before treatment with transglutaminase. The heat treatment can be carried out so that more than 50%, preferably more than 60%, more than 70% or more than 80% of the whey protein in the milk substrate is denatured. In the context of the present invention, the whey protein is denatured when it sediments at pH 4.5. In a more preferred embodiment, the milk substrate is subjected to heat treatment followed by homogenization before treatment with transglutaminase. Optionally, the fermented milk substrate can be subjected to heat treatment, such as pasteurization, to inactivate the microorganism. Another heat treatment can be performed after the enzymatic treatment so that the enzyme is inactivated.

In another preferred embodiment, yeast extract or a reducing agent such as glutathione is added to the milk substrate before treatment with transglutaminase.

Other heat treatment, such as pasteurization, can be carried out after the enzymatic treatment so that the enzyme is inactivated.

The enzyme having transglutaminase activity is added in a suitable amount to achieve the desired degree of protein modification under the chosen reaction conditions. The enzyme can be added at a concentration of between 0.0001 and 1 g / l of milk substrate, preferably between 0.001 and 0.1 g / l of milk substrate. By dosing in units, the enzyme can be added at a concentration of between 0.5 TGHU (transglutaminase hydroxamate units) and TGHU of TGase / g protein in the milk substrate, preferably between 2 and 10 TGHU of TGase / g of protein in the milk substrate.

In another preferred embodiment, yeast extract or a reducing agent such as glutathione is added to the milk substrate before treatment with transglutaminase.

The enzymatic treatment in the methods of the invention can be carried out by adding the enzyme to the milk substrate and allowing the enzymatic reaction to take place during an appropriate occupation time at an appropriate temperature. The enzymatic treatment can be carried out under the conditions chosen to adapt to the protein modifying enzyme selected according to principles well known in the art. The treatment can also be carried out by contacting the milk substrate with an enzyme that has been immobilized.

The enzymatic treatment can be carried out at any suitable pH, such as, for example, in the range of pH 2 10, such as at a pH of 4-9 or 5-7. It may be preferred to let the enzyme act at the natural pH of the milk substrate or, if acidification is obtained due to fermentation, the enzyme may act at the natural pH of the milk substrate during the fermentation process, ie, the pH will gradually decrease from the pH of the unfermented milk substrate to the pH of the fermented milk substrate.

The enzymatic treatment can be carried out at any appropriate temperature, for example, in the range 1-80 ° C, such as 2-70 ° C. In an embodiment of the present invention, the enzymatic treatment can preferably be carried out at a temperature in the range 40-50 ° C. In another embodiment, the enzymatic treatment can preferably be carried out at a temperature below 10 ° C.

Optionally, after the enzyme has been allowed to act on the milk substrate, the enzyme protein can be removed, reduced, and / or inactivated by any method known in the art, such as by heat treatment and / or reducing pH.

Optionally, other ingredients may be added to the acidified milk product, such as color; stabilizers, for example, pectin, starch, modified starch, CMC, etc .; or polyunsaturated fatty acids, for example, omega-3 fatty acids. Such ingredients can be added at any point during the production process, ie, before or after fermentation, before or after enzyme treatment, and before or after the optional addition of syrup.

In the context of the present invention, an enzyme having transglutaminase activity can be an enzyme that catalyzes the transfer of acyl between the gamma carboxylamide group of peptide-linked glutamine (acyl donor) and primary amines (acyl acceptor), for example , peptide-linked lysine. The acid amides and the free amino acids also react. The proteins and peptides can therefore be crosslinked in this way. The transglutaminase can also, for example, if the amines are absent, catalyze the deanimation of glutamine residues in proteins with H2O as the acyl acceptor.

A transglutaminase can also be referred to as, for example, glutamine-gamma-glutamyl transferase protein, factor XlIIa, fibrinoligase, fibrin stabilizing factor, glutamyl peptide gamma-glutamyltransferase, polyamine transglutaminase, tissue transglutaminase, or R-glutaminyl-peptide: gamma-amino glutamyl transferase The group of transglutaminases comprises, but is not limited to, the enzymes assigned to subclass EC 2.3.2.13. In the context of the present invention, transglutaminase can also be referred to as TGase or TG.

A transglutaminase to be used according to the invention is preferably purified. The term "purified" as used herein covers enzyme protein preparations where the preparation has been enriched for the enzyme protein in question. Such enrichment could, for example, be: the elimination of the cells of the organism from which the enzyme protein was produced, the removal of the non-protein material by a specific precipitation of protein or the use of a chromatographic procedure where the enzyme protein in question selectively it is adsorbed and eluted from a chromatographic matrix. The transglutaminase may have been purified to a degree so that only minor amounts of other proteins are present. The term "other proteins" refers in particular to other enzymes. A transglutaminase to be used in the method of the invention may be "substantially pure", that is, substantially free of other components of the organism in which it has been produced, which may be either a natural microorganism or a genetically host microorganism. modified for the recombinant production of transglutaminase. However, for the uses according to the invention, the transglutaminase does not need to be so pure. You can, for example, include other enzymes.

In a preferred aspect, the transglutaminase to be used in the method of the invention has been purified to contain at least 20%, preferably at least 30%, at least 40% or at least 50% (p / p) of transglutaminase of the total protein. The amount of transglutaminase can be calculated from a measure of activity of the preparation divided by the specific activity of the transglutaminase (activity / mg of EP), or it can be quantified by SDS-PAGE or any other method known in the art. The amount of total protein can, for example, be measured by amino acid analysis.

In one embodiment of the methods of the invention, the enzyme having transglutaminase activity is produced recombinantly.

In other embodiments of the present invention, the enzyme having transglutaminase activity may be of animal, plant or microbial origin. Preferred enzymes are obtained from microbial sources, in particular from filamentous fungi or yeasts, or from a bacterium. For the purposes of the present invention, the term "obtained from" as used herein in relation to a given source means that the enzyme originates from the source. The enzyme can be produced from the source or from a strain in which the nucleotide sequence encoding the enzyme has been inserted, i.e., a recombinant strain. In a preferred embodiment, the polypeptide obtained from a certain source is secreted extracellularly.

The enzyme can be obtained, for example, from a strain of Agaricus, for example, A. bisporus; Ascovaginospora; Aspergillus, for example, A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; Chaetomium; Chaetotomastia; Dictyostelium, for example, D. discoideum; Mucor, for example, M. javanicus, M. mucedo, M. subtilissimus; Neurospora, for example, N. crassa; Rhizomucor, for example, R. pusillus; Rhizopus, for example, R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, for example, S. libertiana; Trichophyton, for example, T. rubrum; Whetzelinia, for example, W. sclerotiorum; Bacillus, for example, B. megaterium, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Chryseobacterium; Citrobacter, for example, C. freundii; Enterobacter, for example, E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, for example, E. herbicola; Escherichia, for example, E. coli; Klebsiella, for example, K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, for example, N. crassa; Phytophthora, for example, P. cactorum; Proteus, for example, P. vulgaris; Providence, for example, P. stuartii; Pycnoporus, for example, Pycnoporus cinnabarinus, Pycnoporus sanguineus; Salmonella, for example, S. typhimurium; Serratia, for example, S. liquefasciens, S. marcescens; Shigella, for example, S. flexneri; Streptomyces, for example, S. antibioticus, S. castaneoglobisporus, S. lydicus, S. mobaraensis, S. violeceoruber; Streptoverticilium, for example, S. mobaraensis; Trametes; Trichoderma, for example, T. reesei, T. viride; Yersinia, for example, Y. enterocolitica.

In a preferred embodiment, the enzyme is a transglutaminase obtained from a bacterium, for example, an Actinobacteria of the Actinobacteria class, such as from the subclass Actinobacteridae, such as of the order Actinomycetales, such as from the suborder Streptomycineae, such as from the Streptomycetaceae family, such as from a strain of Streptomyces, such as S. lydicus or S. mobaraesis . In another embodiment, the enzyme is a transglutaminase obtained from a fungus, for example, of the class Oomycetes, such as of the order Peronosporales, such as from the family Pythiaceae, such as from the genera Pythium or Phytophthora, such as from a strain of Phytophthora cactorum .

According to the present invention, the transglutaminase activity can be determined by any method known in the art, such as by incubating the enzyme with gamma carboxamide group of glutamine linked to protein or peptide and an amino group, for example, lysine bound to protein or peptide, in a buffer at various pH and temperatures, for example, 50 mM MES at pH 6.5 at 37 ° C for 30 minutes. The detection of enzyme activity can be followed by the release of ammonia (eg, kit obtained from Roche NH3-11877984) or by using hydroxylamine as an amino group donor (the amount of glutamic acid gamma hydroxamate formed in the reaction is detected as a red complex with ferric ions under acidic conditions measured at 510 nm) or by the determination of epsilon- (gammaglutamyl) lysine by amino acid analysis.

References

WO09016257A (Novozymes); US2009061046A (NovoZymes); WO9421129A (Novozymes); WO2007 / 060288A, WO05016027A; WO0110232A; EP0671885; US4289789

Food Control, Volume 16, Number 3, March 2005, Pages 205-209

Journal of Dairy Science, Vol. 85, No. 7, 1705-1708

Journal of Dairy Research (2008) 75450-456

International Dairy Journal, Volume 16, Number 2, February 2006, Pages 111-118

All references cited in this patent document are incorporated herein by reference in their entirety.

Drawing

Figure 1 represents the gel firmness as a function of the TGase dose, see example.

Example

Yogurt was made from 3 X 200 ml of whole milk:

Sample 1: Whole milk with the 2% added skimmed milk powder (LDP).

Sample 2: Whole milk with the 1% added LDP and 0.2 units of TGase per gram of substrate protein (U / g). Sample 3: Whole milk with the 1% added LDP and 0.5 U / g of TGase.

In all cases, fresh whole milk from Arla Foods (Arla Ekspress, "S0d m ^ lk") was used. Skimmed milk powder was added to the milk and allowed to hydrate overnight at 5C. The milk was then heat treated at 90 ° C for 20 minutes and cooled to the fermentation temperature of 43 ° C. After cooling to 43 C, the necessary amount of TGase (Ajinomoto Activa YG) was added to the milk. After that the culture was added (YL-F800, Chr. Hansen A / S) and the milk was incubated at 43C until a pH of 4.7 was reached. Then the sample was cooled to 5C and stored to measure. The next day the rheological properties of the yogurt were measured using a StressTech rheometer. The measurements were made at 13C. In one test the gel firmness of the yogurt was measured by a so-called frequency sweep, which measures the complex modulus (G *) of the gel as a function of the oscillation frequency. The G * value at 1 Hz was used as the "gel firmness" of the yogurt. The viscosity of the yogurt was measured by a so-called constant speed measurement, which measures the cutting tension of the yogurt as a function of the cutting speed. It has previously been found that the cutting tension at a speed of 300 s-1 correlates well with the perceived sensory "thickness in the mouth", and therefore this value was used to evaluate the "mouth thickness" of the yoghurts. The area that forms between the cut-off voltage curve, when the cutting speed is increased and decreased again frequently is termed as the "hysteresis loop". It has previously been found that the ratio of the area of the "hysteresis loop" divided by the shear stress to 300 s-1 is a measure of the fibrousness of the yogurt, and therefore this value (the ratio) was used to describe the fibrosity of yoghurts.

Figure 1 clearly shows that when TGase is added to yogurt, even when replacing 1% of LDP, the gel firmness of the yogurt significantly improves (as a function of the TGase dose).

The following table (Table 1) shows the cutting tension at 300 s-1 ("Thickness in the mouth") and the fibrosity (measured as described). It is clear that the "thickness in the mouth" of yogurt increases, while the fibrousness decreases. This shows that the TGase together with a culture that produces EPS can produce a yogurt with high gel firmness, high "thickness in the mouth" and a firmer texture, than the one anticipated of using the culture that produces EPS are TGase.

Table 1

Claims (15)

1. A method for producing a yogurt with decreased fibrosity, wherein said method comprises: a) providing a milk substrate; b) treating the milk substrate with an enzyme having transglutaminase activity; Y c) fermenting the milk substrate with a starter culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce the yogurt, wherein said starter culture produces an exopolysaccharide (EPS). 2. The method of claim 1, wherein step b) is performed before or during step c). 3. The method of claim 1, wherein the milk substrate is pasteurized prior to acidification and the enzyme treatment is carried out prior to pasteurization. 4. The method of any of claims 1-3, wherein glutathione is added to the milk substrate prior to treatment with the enzyme having transglutaminase activity. 5. The method of any of claims 1-4, wherein the yogurt is selected from the group consisting of: a firm-type yoghurt, a drinkable yoghurt, and a yoghurt that can be picked up with a spoon. 6. The method of any of claims 1-5, wherein the yogurt has a non-fatty solid milk content of less than 8%. The method of claim 6, wherein the yogurt has a fat content of less than 2%. The method of claim 7, wherein the yogurt has a fat content of less than 0.5%. The method of any of claims 1-8, wherein the enzyme having transglutaminase activity is produced recombinantly. 10. The method of any of claims 1-9, wherein the enzyme having transglutaminase activity is obtained from a bacterium belonging to the genus Streptomyces. The method of any of claims 1-10, wherein the yogurt is produced without any addition of a thickener and / or stabilizer selected from the group consisting of pectin, gelatin, starch, modified starch, carrageenan, alginate and guar gum . The method of any of claims 1-11, wherein a protein hydrolyzate is added to the milk substrate prior to treatment with the enzyme having transglutaminase activity. 13. Use of transglutaminase in combination with a microorganism that produces an exopolysaccharide (EPS) to decrease the fibrosity of an acidified milk product. 14. The use of claim 13, wherein said acidified milk product is a yogurt. 15. The use of claim 14, wherein said yogurt is a firm-type yogurt or can be taken with a spoon.